PROJECT SUMMARY 8.5 million people in the United States suffer from peripheral arterial disease (PAD). As the disease progresses, it can lead to severe obstruction of arterial blood flow to the extremities causing critical limb ischemia, and is associated with devastatingly high mortality rates of up to 20% just 6 months from initial diagnosis. This condition requires immediate endovascular treatment to re-establish blood flow, commonly through the use of stents, balloon angioplasty, or autologous vein grafts; however, these treatments require multiple interventions and do not conclusively lower the amputation rates. Therapeutic interventions aimed at long-term functional recovery must augmenting tissue angiogenesis concomitant with restoring physiological tissue architecture. This K99/R00 Pathway to Independence Award builds on previous work that demonstrates that spatial patterning cues from nanoscale extracellular matrices modulate endothelial cell (EC) morphology and angiogenic function. The objective of the current study is to use nanoscale cell guidance from aligned 3D scaffolds to enhance the angiogenic potential of vascular ECs, with the regenerative goal of restoring blood flow to ischemic regions and enabling functional repair of severely damaged tissue, an important public health goal that has been challenging to attain. First, this award will provide the opportunity to examine the role of spatial patterning using aligned versus non-patterned scaffolds, in the enhancement of EC angiogenic function as well as the modulation of muscle myoblasts phenotype and mechanical properties. In parallel with this aim, the therapeutic efficacy of EC-seeded aligned scaffolds in comparison to non-patterned scaffolds, will be assessed for tissue revascularization and muscle regeneration in a mouse model of volumetric muscle and vascular injury. Through these studies, the challenge of restoring both vascular and muscular function to injured tissues is tackled on multiple fronts by using spatial cell patterning to induce an EC phenotype concomitant with angiogenesis that will in turn enhance muscle myofiber differentiation and maturation. Finally, to gain a deeper understanding of the mechanisms by which gene networks and pathways work in concert to promote angiogenesis through spatial patterning, methodologies in gene silencing and functional genomics will be employed to reveal novel cell patterning pathways. The proposed training will include courses offered through the Stanford School of Medicine and externships with leading experts in the fields of cardiovascular medicine, data science, and muscle regeneration. The proposed series of studies will deepen the understanding of the biological mechanisms through which spatial cell patterning confers enhancement of EC angiogenesis and muscle myoblast function. Findings from these studies will provide insights that will inform future regenerative strategies and engineered therapeutics for revascularization of severely damaged and ischemic tissues, and will serve as an innovative platform and important step in the treatment of a broad range of vascular diseases.